Peptides for preventing or treating liver damage

ABSTRACT

The application of a peptide having sequence of formula I or its derivant in preparing the medicine for preventing or treating liver damage, especially liver damage and hepatitis C is disclosed, Xaa1-Gln-Xaa2-Xaa3-Thr-Ser-Gly-Xaa4 (formula I) wherein, Xaa1 is deletion, Ala, Gly, Val, Leu or Ile, Xaa2 is Thr or Ser, Xaa3 is Tyr, Phe or Trp, and Xaa4 is deletion, Ala, Gly, Val, Leu, Ile or Pro. The composite medicine containing the said peptide, its preparation method, and the polynucleotide for coding the said peptide are also disclosed.

FIELD OF THE INVENTION

The present invention relates to the use of an immunogenic peptide ofHepatitis C virus or a derivative thereof in preventing or treatingliver damage, preferably relates to the use of the peptide or aderivative thereof in preventing or treating immunological liver damageor hepatotoxic chemical substance-induced liver damage, and to the usein preventing or treating Hepatitis C. The present invention alsorelates to a pharmaceutical composition comprising the peptide or aderivative thereof, a method of manufacturing the peptide and apolynucleotide encoding the peptide.

BACKGROUND OF THE INVENTION

Viral hepatitis is a group of severe diseases that do harm to humanhealth, the causative agents of which are a group of differenthepatovirus. There are 7 types of hepatitis virus found hitherto, whichare HAV, HBV, HCV, HDV, HEV and possible TTV and HGV. Among them,Hepatitis C virus (HCV) is a causative agent that results in HepatitisC. Though Hepatitis C was originally identified as non-A, non-Bhepatitis acquired by transfusion, the subsequent research showed thatHepatitis C virus is propagated not only by transfusion, but also byother ways via alimentary tract, sex act and so on. At present there areapproximately more than 100 million persons infected with HCV in theworld, and approximately 50%-90% of those persons will develop chronicdisease. 8-46% and 11-19% from those with chronic disease will furtherdevelop hepatocirrhosis and hepatocellular carcinoma respectively.

HCV is a kind of RNA virus of Flavividae family. The studies (see, e.g.,Choo et al, Science 244:359-362 (1989); Choo et al, Proc. Natl. Acad.Sci. USA 88:2451-2455 (1991); Han et al, Proc. Natl. Acad. Sci. USA 88:1711-1715 (1991)) indicated that the HCV genome is a single positive RNAstrand of about 9.4 kb and has an open reading frame (ORF) that nearlyspans the whole genome. The ORF encodes a viral polyprotein precursor of3011 or 3010 amino acids. The proteins encoded by the HCV genome includethe nucleocapsid core protein (C), the two envelope glycoproteins (E1and E2), and the genome contains five regions corresponding tononstructural proteins (NS1˜NS5). In the genome, the hypervariableregion 1 (HVR1) from E2 region of Hepatitis C virus contains importantantigenic epitopes that can induce neutralizing antibodies (see, e.g.,Shirai et al, J. Immunol, 162:568-576 (1999)). However, the gene of HVR1may be greatly mutated due to immunological selection so that HCV canescape from the recognition of the body immune system. This might be themajor cause whereby HCV results in chronic hepatitis.

At present, the mechanism, whereby HCV causes disease is not wellunderstood, and there are no very clinically effective therapeuticmethods and vaccines to prevent its propagation. Interferon (IFN) isoften used in the current clinical therapy to degrade viral RNA byactivating RNase L, but the long-term effective rate of the therapy isonly about 20%. Manns et al (The Lancet, 358: 958-965 (2001)) treatedHepatitis C by using both of pegylated interferon and ribavirin. Whilethis therapy is particularly effective in the case of patients infectedby viral strains belonging to genotypes 2 and 3, it only has a limitedeffect on genotypes 1a, 1b and 4. Therefore, people have tried by avariety of means to research and develop vaccines that decrease HCVinfection and medicines that cure hepatitis.

From the study of the HCV genome, people have found a number ofimmunogenic peptides against HCV, which can induce an immune response toHCV in the body. For example, U.S. Pat. No. 5,709,995A of Chisari et aldiscloses a group of peptides that stimulate HCV-specific cytotoxic Tlymphocyte (CU). WO2003/097677A discloses HCV antigenic peptides andcompositions thereof, which have the ability to induce strong immuneresponses. CN1194986C and CN1216075C of the inventor of the presentinvention also disclose a group of HCV immunogenic peptides that caninduce the generation of antibodies. Although it was thought in theearly time that HCV caused liver damage by virus-induced cytopathy likecertain viruses (e.g., Hepatitis C virus, EB virus), the recent studiesindicated that the immune response against HCV is the major causeleading to liver damage. Particularly in chronic patients with HCV, thelymphocyte (especially cytotoxic T lymphocyte) can not completelyeffectively eliminate HCV, but in the process of eliminating hepatocytesinfected with HCV, it results in immunological hepatocyte damage, andthus leads to hepatocyte apoptosis and even to hepatocirrhosis andhepatocellular carcinoma (see, for example, Nelson et al, J. Immunol.,158:1473-1481 (1997); Wong et al, J. Immunol., 160:1479-1488 (1998);Ruggieri et al., Virology, 229 : 68-76 (1997)). Therefore, the HCVimmunogenic peptide used as vaccine will induce an immune responseagainst HCV that might lead to immunological liver damage. Practically,such mechanism greatly impedes the development of prophylactic vaccineor therapeutical medicine for Hepatitis C (especially chronic HepatitisC) from such HCV immunogenic peptides.

Except immunological liver damage and pathogenic liver damage, it iswell known that hepatotoxic chemical substance can also induce liverdamage. It is known that some drugs can cause liver damage, and resultin hepatic cytolysis and necrosis. For example, the analgeticacetaminophen (i.e., Panadol, the chemical name of which is4-(N-acetylamino) phenol), when administrated in a large dose, is a kindof liver-damaging substance that can induce necrosis of human liver. Forexample, long-term administration of antibiotic, such as rifampicin,pyrazinamide, and isoniazide, and long-term administration of estrogenand the like in the period of menopause, also can cause severehepatocyte necrosis, leading to liver damage, such as acute or chronichepatitis, jaundice, and hepatic fibrosis and the like. Theliver-damaging substances include substance that can generate manyactive free radicals, particularly include substance that can generateoxygen-derived free radicals, which induce hepatotoxity via oxidation.

After massive research, the inventor obtained an HCV immunogenic peptideand derivative thereof that can prevent or treat liver damage, andsurprisingly, the said liver damage is not limited to immunologicaldamage of hepatocyte infected with HCV. For example, the peptide andderivative thereof of the invention can be used to prevent or treatimmunological liver damage, pathogenic liver damage and hepatotoxicchemical substance-induced liver damage.

DESCRIPTION OF THE INVENTION

The invention relates to a peptide and derivative thereof, which can beused for preventing or treating liver damage in addition to inducementof immune response against HCV, particularly for preventing or treatingimmunological liver damage, wherein the said liver damage is not limitedto immunological damage of hepatocyte infected with HCV.

In the first aspect, the invention provides a use of a peptide havingthe sequence of Formula I or a pharmaceutically acceptable salt or esterthereof,

Xaa1-Gln-Xaa2-Xaa3-Thr-Ser-Gly-Xaa4  (Formula I)

wherein,

Xaa1 is absent, Ala, Gly, Val, Leu or Ile,

Xaa2 is Thr or Ser,

Xaa3 is Tyr, Phe or Trp, and

Xaa4 is absent, Ala, Gly, Val, Leu, Ile or Pro,

and the said use is for preventing or treating liver damage or for themanufacture of a medicament for preventing or treating liver damage.Namely, the invention provides a use of a peptide having the sequence ofFormula I or a pharmaceutically acceptable salt or ester thereof inpreventing or treating liver damage, and provides a use of a peptidehaving the sequence of Formula I or a pharmaceutically acceptable saltor ester thereof in the manufacture of a medicament for preventing ortreating liver damage.

The phrase “liver damage”, when used herein, refers to injury orpathological change present in liver tissue or cell. The clinicalconditions of liver damage include degeneration of live cells,vasculitis of liver, spotty necrosis or focal necrosis present in liver,inflammatory cell infiltration or fibroblast proliferation in liver andportal area, or hepatomegaly, and hepatocirrhosis, hepatoma resultedfrom severe liver damage, and the like. For evaluation of liver damage,in addition to pathological conditions as mentioned above, determinationof aminotransferase activity in serum can be used to diagnose the damageof liver and to assess the degree of damage, because due to cytolysis ofdamaged liver cells, the amount of the circulating aminotransferasereleased from damaged liver cells increases. In the embodiment of theinvention, the liver damage can be determined by measuring the level ofglutamic-pyruvic transaminase or glutamic-oxalacetic transaminase.Preferably in the invention, the liver damage can be liver damageindicated by the level of glutamic-pyruvic transaminase orglutamic-oxalacetic transaminase in serum, and the indication ofcurative effect on prevention or treatment of liver damage can be thedecrease in the level of glutamic-pyruvic transaminase orglutamic-oxalacetic transaminase. The effect of the peptide having thesequence of Formula I of the invention or a pharmaceutically acceptablesalt or ester thereof on liver damage can be determined from thealleviation of pathological conditions as mentioned above and thedecrease in the level of glutamic-pyruvic transaminase orglutamic-oxalacetic transaminase.

Current study has found that a variety of hepatotoxic drugs and chemicalsubstance, immunogen or pathogen can cause liver damage. Preferably theliver damage as mentioned in the invention is immunological liver damageor hepatotoxic chemical substance-induced liver damage. Namely, theinvention preferably provides a use of a peptide having the sequence ofFormula I or a pharmaceutically acceptable salt or ester thereof inpreventing or treating immunological liver damage or hepatotoxicchemical substance-induced liver damage, and preferably provides a useof a peptide having the sequence of Formula I or a pharmaceuticallyacceptable salt or ester thereof in the manufacture of a medicament forpreventing or treating immunological liver damage or hepatotoxicchemical substance-induced liver damage. At present, there are a varietyof animal models used to assess the effect on liver damage in theinvention. In a specific example, the peptide of the invention canalleviate immunological liver damage induced by BCG vaccine andlipopolysaccharide. In addition, in a specific model of hepatotoxicchemical substance-induced liver damage, the peptide of the inventioncan prevent liver damage induced by D-aminogalactose, and the peptide ofthe invention can also treat liver damage induced by tetrachloromethane.

A “peptide having the sequence of Formula I” used in the invention,refers to a peptide of the sequence of Formula I or a modifiedfunctional equivalent thereof. Namely, the peptide of the sequence ofFormula I may not be modified at its amino group at N-terminus, carboxylgroup at C-terminus and groups at side chains of amino acids therein, ormay be modified without substantially diminishing its effect onprevention or treatment of liver damage. A “functional equivalent” usedherein refers to a modified product comprising the sequence of FormulaI, of which the effect on prevention or treatment of liver damage is notsubstantially diminished, namely the effect of prevention or treatmentof liver damage is diminished by less than 50%, preferably by less than30%, more preferably by less than 10%, and most preferably notdiminished. Some pathological conditions or levels of someaminotransferases in serum can be used to determine the effect of afunctional equivalent on prevention or treatment of liver damage,preferably the pathology score criterion or the level ofglutamic-pyruvic transaminase or glutamic-oxalacetic transaminase inserum as mentioned in the embodiment of the invention is used todetermine the effect.

For the peptide of the invention, suitable modifications include, forexample, cyclization, polymerization, modification at the terminal aminogroup, carboxyl group or side chain groups to form a pharmaceuticallyacceptable ester, conjugation to form a conjugate comprising thesequence of Formula I, fusion to form a fusion protein comprising thesequence of Formula I, or combinations thereof, etc. In general, alinear peptide can be cyclized, for example, by linking the N-terminalamino group of the peptide to the C-terminal carboxyl group of thepeptide to form a cyclic peptide, to prolong its half-life in thephysiological environment. A “pharmaceutically acceptable ester” refersto an ester suitable for contact with human or animal tissues withoutleading to various toxicity, stimulation or allergy, etc. Generally,esterification of a peptide can reduce proteolysis by proteases in thebody. The peptide of the invention can be modified at the terminal aminogroup, carboxyl group or side chain groups to form a pharmaceuticallyacceptable ester. Modifications at side chain groups include, but arenot limited to, esterification between the side chain group of threonineor serine and carboxylic acid. Modifications at N-terminal amino groupinclude, but are not limited to, deamination, and modification withN-lower alkyl, N-di-lower alkyl and N-acyl. Modifications at C-terminalcarboxyl group include, but are not limited to, modification to beamide, lower alkyl amide, dialkyl amide, and lower alkyl ester.Preferably, the terminal groups are protected by protective groups knownto a skilled person in the field of protein chemistry, such as acetyl,trifluoroacetyl, Fmoc (9-fluorenyl-methoxycarbonyl), Boc (teriarybutoxycarbonyl), Alloc (allyloxycarbonyl), C₁₋₆ alkyl, C₂₋₈ alkenyl,C₇₋₉ aralkyl, etc. In an embodiment of the invention, preferably thepeptide of Formula I is not modified at N-terminal amino group,C-terminal carboxyl group and side chain groups of amino acids therein,namely the group at N-terminus remains α-amino group (—NH₂) of the firstamino acid, the group at C-terminus remains carboxyl group (—COOH) ofthe C-terminal amino acid. Preferably in the invention, C-terminalcarboxyl group can also be amidated, namely the group at C-terminus is—CONH₂.

Using methods known in the art, a conjugate comprising the sequence ofFormula I may comprise a pharmaceutically acceptable water-solublepolymer moiety. In general, such conjugate can be shown to enhance thecirculating half-life of the peptide of the sequence of Formula I.Suitable water-soluble polymers include polyethylene glycol (PEG),monomethoxy-PEG, mono-(C_(1-C10))alkoxy-PEG, aryloxy-PEG, poly-(N-vinylpyrrolidone)PEG, trimethoxy PEG, monomethoxy-PEG propionaldehyde, PEGpropionaldehyde, disuccinimidyl carbonate PEG, propylene glycolhomopolymers, a polypropylene oxide/ethylene oxide co-polymer,polyoxyethylated polyols (e.g., glycerol), monomethoxy-PEGbutyraldehyde, PEG butyraldehyde, monomethoxy-PEG acetaldehyde, PEGacetaldehyde, methoxyl PEG-succinimidyl propionate, methoxylPEG-succinimidyl butanoate, polyvinyl alcohol, dextran, cellulose, orother carbohydrate-based polymers. Suitable PEG may have a molecularweight from about 600 to about 60,000, including, for example, 5,000daltons, 12,000 daltons, 20,000 daltons, 30,000 daltons, and 40,000daltons, which can be linear or branched. A conjugate comprising thesequence of Formula I can also comprise a mixture of such water-solublepolymers. Pegylation can be carried out by any of the PEGylationreactions known in the art (see, for example, Delgado et al, CriticalReviews in Therapeutic Drug Carrier Systems 9:249 (1992), Duncan andSpreafico, Clin. Pharmacokinet. 27:290 (1994), and Francis et al, Int JHematol 68: 1 (1998)). For example, Pegylation can be performed by anacylation reaction or by an alkylation reaction with a reactivepolyethylene glycol molecule. In an alternative approach, a conjugateare formed by condensing activated PEG, in which a terminal hydroxy oramino group of PEG has been replaced by an activated linker (see, forexample, Karasiewicz et al, U.S. Pat. No. 5,382,657A). A conjugatecomprising the sequence of Formula I also may be a conjugate formed bythe couple of the peptide of the sequence of Formula I to otherproteins. The said other proteins include human albumin, bovine albumin,or Fc portion of IgG molecule. In an embodiment of the invention, thepeptide of the invention is coupled to bovine albumin to form a peptideconjugate.

A peptide having the sequence of Formula I of the invention also may bea fusion peptide or fusion protein comprising the sequence of Formula I,which is formed by the peptide of the sequence of Formula I fused toother peptide or protein. The said other protein may be human albumin,bovine albumin, or Fc portion of IgG molecule. Albumin can begenetically coupled a peptide having the sequence of Formula I of theinvention to prolong its half-life. And human albumin is the mostprevalent naturally occurring blood protein in the human circulatorysystem, persisting in circulation in the body for over 20 days. Researchhas shown that therapeutic proteins genetically fused to human albuminhave longer half-lives. And other research has shown that the resultantfusion protein fused with Fc portion may have an increased circulatinghalf-life (See, U.S. Pat. No. 5,750,375A, U.S. Pat. No. 5,843,725A, U.S.Pat. No. 6,291,646; Barouch et al, Journal of Immunology, 61:1875-1882(1998); Barouch et al, Proc. Natl. Acad. Sci. USA, 97(8):4192-4197 (Apr.11, 2000); and Kim et al, Transplant Proc, 30(8):4031-4036 (December1998)).

In the peptide having the sequence of Formula I of the invention,preferably Xaa1 is Gly, Xaa2 is Thr, Xaa3 is Tyr, and Xaa4 is absent,Ala or Gly, more preferably Xaa4 is absent. “Absent” used herein refersto the absence of the absent amino acid residue in the peptide sequence.For example, when Xaa4 is absent, the amino acid at C-terminus of thesequence of Formula I is Gly in Formula I.

All symbols of the peptide, amino acid, and group used herein are wellknown in the art. The abbreviations of amino acids or amino acidresidues are defined in Table 1, and these abbreviations may representL-amino acids, or may represent D-amino acids, preferably representL-amino acids. The amino acids or amino acid residues can be dividedaccording to the similarity of properties of amino acid side chains intothe following groups: hydrophobic amino acids (A, I, L, M, F, P, W, Y,V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), aminoacids having an aliphatic side chain (G, A, V, L, I, P), amino acidshaving a hydroxyl group-containing side chain (S, T, Y), amino acidshaving a sulfur-containing side chain (C, M), amino acids having acarboxylic acid- or amide-containing side chain (D, N, E, Q), aminoacids having a base-containing side chain (R, K, H), and amino acidshaving an aromatic-containing side chain (H, F, Y, W). The amino acidsor amino acid residues within the same group generally have similarproperties.

TABLE 1 the Abbreviations of Amino Acids Amino Acid One Letter CodeThree Letter Code alanine Ala A arginine Arg R asparagine Asn N asparticacid Asp D cysteine Cys C glutamine Gln Q glutamic acid Glu E glycineGly G histidine His H isoleucine Ile I leucine Leu L lysine Lys Kmethionine Met M phenylalanine Phe F proline Pro P serine Ser Sthreonine Thr T tryptophan Trp W tyrosine Tyr Y valine Val V

A “pharmaceutically acceptable salt” refers to a salt suitable forcontact with human or animal tissues without leading to varioustoxicity, stimulation or allergy, etc. A pharmaceutically acceptablesalt is well known in the art. Such salt may be prepared in the processof final isolation and purification of the peptide of the invention, ormay be prepared by a further reaction with a suitable organic orinorganic acid or base. The representative acid addition salts include,but are not limited to, acetate, dicaproate, alginate, citrate,aspartate, benzoate, benzenesulphonate, bisulfate, butyrate, camphorate,camphorsulfonate, glycerophosphate, sulfite, heptylate, hexanoate,fumarate, hydrochlorate, hydrobromide, hydriodate, 2-hydroxyethylsulphonate, lactate, maleate, mesylate, nicotinate,2-naphthalenesulfonate, oxalate, 3-phenylpropionate, propionate,succinate, tartrate, phosphate, glutamate, bicarbonate, p-toluenesulfonate, and undecanate. The preferable acids used to formpharmaceutically acceptable salts arehydrochloric acid, hydrobromicacid, sulfuric acid, phosphoric acid, oxalic acid, maleic acid, succinicacid, and citric acid. The cations in pharmaceutically acceptable baseaddition salts include, but are not limited to, ions of alkali metals oralkaline earth metals such as potassium, calcium, magnesium, andaluminium, and cations of nontoxic quaternary ammoniums such asammonium, tetramethyl ammonium, tetraethyl ammonium, methylamine,dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine,ethanolamine, diethanolamine, piperidine, piperazine, and the like. Thepreferable salts include phosphate, tris, and acetate. Such salts maygenerally increase the solubility of peptides without substantiallyaltering their activity. The peptide of the invention may be utilizeditself, or utilized in the form of pharmaceutically acceptable salt.

The use of the first aspect of the invention may further include the usefor preventing and/or treating Hepatitis C. Namely, in addition to theprevention or treatment of liver damage, the invention further providesa use of a peptide having the sequence of Formula I or apharmaceutically acceptable salt or ester thereof in preventing ortreating liver damage and Hepatitis C simultaneously, and provides a useof a peptide having the sequence of Formula I or a pharmaceuticallyacceptable salt or ester thereof in the manufacture of a medicament forpreventing or treating liver damage liver damage and Hepatitis Csimultaneously. The peptide of the invention has the ability to inducecytokines such as γ-IFN, IL-4, and IL-10, and antibodies. Among them,γ-IFN, a significant cytokine secreted from Class I helper T cell (Th1),is one of important cytokines against virus infection in immune systemof the body, which can induce cellular immune response against HCV toeliminate HCV, and has been used in the current well-established HCVtherapy.

In the second aspect, the invention provides a pharmaceuticalcomposition comprising a peptide having the sequence of Formula I or apharmaceutically acceptable salt or ester thereof and a pharmaceuticallyacceptable carrier,

Xaa1-Gln-Xaa2-Xaa3-Thr-Ser-Gly-Xaa4  (Formula I)

wherein,

Xaa1 is absent, Ala, Gly, Val, Leu or Ile,

Xaa2 is Thr or Ser,

Xaa3 is Tyr, Phe or Trp, and

Xaa4 is absent, Ala, Gly, Val, Leu, Ile or Pro,

for the use according to the first aspect of the invention.

The pharmaceutical composition of the invention may be used to preventor treat liver damage. The pharmaceutical composition of the inventioncan reduce some pathological conditions resulted from liver damageand/or decrease the level of some aminotransferase in serum, andpreferably reduce the pathology score as described in the embodiment ofthe invention or decrease the level of glutamic-pyruvic transaminase orglutamic-oxalacetic transaminase in serum. The pharmaceuticalcomposition of the invention has the ability to induce cytokines such asγ-IFN, IL-4, and IL-10, and antibodies. Thus, preferably thepharmaceutical composition of the invention the invention may be usedfurther to treat and/or prevent Hepatitis C.

A “pharmaceutically acceptable carrier” used herein refers to nontoxicsolid, semisolid, or liquid filler, diluent, adjuvant, coating agent orother supplement material for formulation. Depending upon thetherapeutic purpose and the route of administration, the pharmaceuticalcomposition may be formulated by well-known techniques in the art as avariety of formulations. Such composition may preferably be presented inunit dosage form, such as tablet, film, ovule, capsule (includingsustained-release or delayed-release form), powder, granule, tincture,syrup or emulsion, sterile injectable solution or suspension, aerosol orspray, drop, injection, auto-transfusion device or suppository. Forexample, it may be administered in the form of tablets or capsules. Theactive medical ingredients as mentioned above may be combined with akind of nontoxic pharmaceutically acceptable oral inert carrier, such asethanol, isotonic glucose solution, glycerol, physiological saline, orcombinations thereof. The composition may further comprise othersupplement materials including, for example, protein protection agentssuch as human serum albumin, peptide having low molecular weight, aminoacid, and metal cation, and adjuvants, such as Freund's completeadjuvant, Freund's incomplete adjuvant, and poly(CpG), etc.

However, it is surprising that the pharmaceutical composition of theinvention by itself, without any adjuvants, is capable of preventing ortreating liver damage, and/or of preventing or treating Hepatitis C,wherein the said composition comprises a peptide having the sequence ofFormula I or a pharmaceutically acceptable salt or ester thereof. In oneembodiment, without any adjuvants, the peptide of the invention cansignificantly increase the amount of interferon secreted from the body.In another embodiment, without any adjuvants, the peptide of theinvention can significantly reduce liver damage. These facts suggest,the mechanisms whereby the peptide of the invention prevents or treatsliver damage and Hepatitis C are not limited to the inducement of immuneresponse against HCV. The peptide of the invention, when inhibitingvirus replication and eliminating virus, might further inhibit excessiveimmune inflammatory reaction in the body, and thereby achieve thereduction of the damage of liver tissues and cells. Thus, thecomposition of the second aspect of the invention preferably comprisesno adjuvant.

In the peptide having the sequence of Formula I in the pharmaceuticalcomposition of the invention, preferably Xaa1 is Gly, Xaa2 is Thr, Xaa3is Tyr, and Xaa4 is absent, Ala or Gly, more preferably Xaa4 is absent.

Furthermore, the invention also relates to a use of the pharmaceuticalcomposition of the second aspect of the invention in the manufacture ofa medicament for preventing or treating liver damage; the invention alsorelates to a use of the pharmaceutical composition of the second aspectof the invention in preventing or treating liver damage. Preferably, theuse as mentioned above may further include the use for treating and/orpreventing Hepatitis C.

The pharmaceutical composition of the invention can be administered byany routes well known to a person skilled in the art, for example, oral,rectal, sublingual, intrapulmonary, transdermal, iontophoretic, vaginal,and intranasal administration. Preferably, the pharmaceuticalcomposition of the invention is administered by parenteral routes, suchas subcutaneous, intramuscular or intravenous injection. The dosage tobe administered will vary depending upon the formulation, the desiredtime-course and the subject to be treated, and a physician can readilydetermine the feasible dosage in the therapy based on the practicalsituation (e.g., the condition of the patient, body weight and so on).For a general adult, the dosage of the pharmaceutical composition of theinvention may be 1 ng-10 g of the peptide having the sequence of FormulaI per kg body weight of the adult. For the route of injection, thedosage preferably is 100 ng-10 mg per kg body weight, more preferably 1μg-1 mg per kg, and most preferably 10 μg-100 μg per kg. For the oraladministration, the daily dosage to be administered may be 1 μg-10 g perkg body weight, preferably 10 μg-1 g per kg body weight, and morepreferably 100 μg-10 mg.

In the third aspect, the invention provides a peptide having thesequence of Formula I or a pharmaceutically acceptable salt or esterthereof,

Xaa1-Gln-Xaa2-Xaa3-Thr-Ser-Gly-Xaa4  (Formula I)

wherein,

Xaa1 is absent, Ala, Gly, Val, Leu or Ile,

Xaa2 is Thr or Ser,

Xaa3 is Tyr, Phe or Trp, and

Xaa4 is absent, Ala, Gly, Val, Leu, Ile or Pro, for the use according tothe first aspect of the invention or used in the pharmaceuticalcomposition according to the second aspect of the invention. In theformula, preferably Xaa1 is Gly, Xaa2 is Thr, Xaa3 is Tyr, and Xaa4 isabsent, Ala or Gly, more preferably Xaa4 is absent.

The peptide of the invention is a purified peptide with at least 80%pure, preferably at least 90% pure, more preferably at least 95% pure,and particularly with pharmaceutical purity, i.e. at least 98% pure, andpathogen-free and pyrogen-free. Preferably, the peptide of the inventionmay substantially comprise no other polypeptides or proteins, especiallythose derived from animals.

Furthermore, in the fourth aspect, the invention provides apolynucleotide encoding the peptide according to the third aspect of theinvention. A “polynucleotide” used herein refers to a single stranded ordouble stranded polymer of deoxyribonucleotides or ribonucleotides, withthe sequence reading from 5′ end to 3′ end, including RNA and DNA. Itmay be prepared by isolation from the natural source, in vitrosynthesis, or recombinant expression.

In the fifth aspect, the invention provides a method of manufacturing apeptide or a pharmaceutically acceptable salt or ester thereof accordingto the third aspect of the invention, wherein the method comprises thestep of chemically synthesizing the said peptide or expressing the saidpeptide in the fusion form. The method of manufacturing of the inventionmay further comprise a reaction step for generating the said salt orester from the said peptide.

It is obvious for a skilled person in the art to chemically synthesize apeptide of known structure. For further details, see the followingpublications, for example, for the solid phase peptide synthesis, see J.M. Steward and J. D. Young, Solid Phase Peptide Synthesis, the 2^(nd)Edition, Pierce Chemical Co., Rockford, Ill. (1984), and J. Meienhofer,Hormonal Proteins and Peptides, Volume 2, Academic Press, New York(1973), etc; and for the liquid phase peptide synthesis, see E. Schroderand K. Lubk, The Peptides, Volume 1, Academic Press, New York (1965),etc. In an embodiment of the invention, the peptides of the inventionare synthesized by solid phase synthesis.

It is obvious for a skilled person in the art to express a peptide ofknown structure in the fusion form and to purify it. (See, for example,Sambrook et al, Molecular Cloning: A Laboratory Manual, the 2^(nd)Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989; and Ausubel et al, Ed., Current Protocols in Molecular Biology,JohnWiley and Sons, Inc. NY, 1987). The said peptide may be prepared byintroducing the polynucleotides according to the fifth aspect of theinvention into an expression vector and expressing it by a host cell.Suitable vectors include plasmid, cosmid, phage, or virus, and the like;and suitable host cells include bacterium, fungus, and eukaryotic cell.

DESCRIPTION OF THE DRAWINGS

FIG. 1. The levels of various antibodies in sera of the mice immunizedwith the peptides of the invention.

FIG. 2. The amount of γ-IFN secreted from splenocytes of the ratsinoculated with the peptides of the invention with or without adjuvants.

FIG. 3. The effects on biochemical criteria of sera in the model of BCGvaccine and lipopolysaccharide-induced immunological liver damage of ratafter immunization with the peptides of the invention, in which *indicates that the corresponding group can significantly decrease thelevel of aminotransferase relative to the model group.

FIG. 4. The scores of pathological conditions of liver in the model ofBCG vaccine and lipopolysaccharide-induced immunological liver damage ofrat after immunization with the peptides of the invention, in which *indicates that the corresponding group can significantly alleviate thepathological conditions of liver relative to the model group.

FIG. 5. The effects on biochemical criteria of sera in the model ofD-aminogalactose-induced acute liver damage of rat after immunizationwith the peptides of the invention, in which * indicates that thecorresponding group can significantly decrease the level ofaminotransferase relative to the model group.

FIG. 6. The effects on biochemical criteria of sera in the model oftetrachloromethane-induced liver damage of mouse after immunization withthe peptides of the invention, in which * indicates that thecorresponding group can significantly decrease the level ofaminotransferase relative to the model group.

FIG. 7. The scores of pathological conditions of liver in the model oftetrachloromethane-induced liver damage of mouse after immunization withthe peptides of the invention, in which * indicates that thecorresponding group can significantly alleviate the pathologicalconditions of liver relative to the model group.

The publications cited in the application are used to illustrate theinvention, the contents of which are incorporated herein by reference,as if they have been written down herein.

For a better understanding of the invention, it will now be described ingreater detail by reference to specific Examples. It should be notedthat the examples only exemplify the invention, and should not beconstrued as limiting the scope of the invention. According to thedescription of the application, various modifications and alterations ofthe invention are obvious to a skilled person in the art.

EXAMPLES Example 1 Synthesis of Peptides

Three peptides of the following sequences were synthesized by solidphase peptide synthesis using Model 413A Automatic Peptide Synthesizer(purchased from Perkin Elmer Corporation): GQTYTSG (referred hereinafterto as “Peptide A”), GQTYTSGA (referred hereinafter to as “Peptide B”)and GQTYTSGG (referred hereinafter to as “Peptide C”). All of aminoacids in the three peptides are L-amino acids. The detailed synthesiswas as follows: at first, the reactive groups of the amino acid monomerswere protected by protecting groups, i.e., 9-fluorenylmethoxycarbonyl(Fmoc) groups for the α-amino groups of amino acids, teriarybutyl groupsfor the side chains of Ser and Thr, and trityl (Trt) groups for the sidechains of Gln. Then the coupling of the protected amino acids wasperformed successively withN,N-diisopropylcarbodiimide/1-hydroxylbenzotriazole as coupling agent,for 40 min for each amino acid. In the presence of 15% ofdithioglycol/dimethyl thioether/anisole (1:1:1 of volume), the cleavageof the peptides from the resin substrate was performed withtrifluoroacetic acid (85%) at room temperature for 120 min, while theprotecting groups were removed. Subsequently the resultant peptides wereprecipitated in anhydrous ether, and then washed several times withanhydrous ether, so as to completely remove mercaptan. Afterprecipitating in water/tertbutyl alcohol (1:1) and then freeze-drying,the raw peptides were made. Within 30 min, the raw peptides werepurified by reverse phase HPLC with the gradient of 37-42%acetonitrile/0.9% TFA. After concentrating and then freeze-drying,Peptide A, Peptide B, and Peptide C were made respectively. All of thethree peptides were white solids with at least 98% pure.

Example 2 Preparation of a Peptide Conjugate

Peptide A was conjugated to bovine serum albumin (BSA) by glutaraldehydeto generate a conjugate. The detailed conjugation process was asfollows: 1 mg of Peptide A synthesized in Example 1 was dissolved into0.5 ml PBS (pH7.4, 0.02 mol/L); and 4.5 mg of BSA was dissolved into 4.5ml PBS (pH7.4, 0.02 mol/L). The resultant Peptide A solution was mixedwith the BSA solution, and then added gently to 1 ml of 0.1%glutaraldehyde. The conjugation reaction was performed for 12 hours atroom temperature in the dark. Then glycine solution (1 mol/L) was addedgently to quench the reaction, before dialysis into PBS (pH7.4, 0.02mol/L) overnight, and then freeze-drying. The resultant Peptide A-BSAconjugate product was referred to as Peptide Conjugate A.

Example 3 Reactivity of the Peptides to Sera of Patients with HepatitisC

3.1 Sources of Sera

Anti-HCV antibody-positive sera: randomly obtained from inpatientsinfected with HCV in the Liberation Army 302 Hospital from 2000 to 2001.

Control sera: obtained from healthy blood donors, who had normal bloodindexes in each assay.

Sera of patients with Hepatitis B (HBV): obtained from inpatients withHepatitis B in the Liberation Army 302 Hospital, who was positive forthe surface antigen of Hepatitis B virus as assayed.

3.2 Method of Determining Reactivity to Sera by Indirect ELISA Assay

The reactivity of the individual peptide of the invention to sera wasdetected by standard indirect ELISA technology using Model DG3022Enzyme-Linked Immunoassay Analyzer (purchased from Perkin ElmerCorporation). 10 μg of Peptide A, Peptide B, Peptide C, and PeptideConjugate A were added respectively into the basic coating buffer (0.1mol/L NaHCO₃, 35 μl; 0.1 mol/L Na₂CO₃, 15 μl; H₂O, 50 μl), and thentransferred into the wells of polypropylene/ethylene microtiter plate,and placed at 4° C. overnight. After adding 120 μl of FCS-PBS, each wellwas blocked at 37° C. for 2 hours. Sera (dilution of 1:100) were thenadded to the wells, and incubated at 37° C. for 1 hour. Goat anti-humanIgGs (dilution of 1:1000) (purchased from Sino-American BiotechnologyCompany, Beijing) were then added to the wells, and incubated at 37° C.for 1 hour. 50 μl of Solution A and Solution B (purchased from BeijingKewei Reagent Company) were added to every well, placed in the dark for5 min, and then the absorbance at 450 nm was measured.

3.3 Results

All of the peptides (Peptide A, Peptide B, Peptide C, and PeptideConjugate A) are negative for sera of 10 patients with HBV and for seraof 10 healthy donors in ELISA Assay. The results of reactivity of thepeptides to anti-HCV antibody-positive sera (of 30 patients) are shownin Table 2. As calculated by X²-test of SATA software, when compared tothe reactivity to sera of patients with HBV and healthy donors, PeptideA, Peptide B, Peptide C, and Peptide Conjugate A of the invention cansignificantly react to sera of patients with HCV, and the conjugate ofcoupling the peptide to bovine serum albumin results in the increase inthe reactivity of the peptide of the invention to sera of patients withHCV.

TABLE 2 Reactivity of the Peptides of the Invention to Sera of Patientswith HCV The Number of Percentage of Peptide Positive Cases PositiveCases (%) Peptide A 13 43 Peptide B 15 50 Peptide C 14 47 PeptideConjugate A 23 77

Example 4 Study on In Vivo Immunogenicity of Peptides and Cytokines inSera in Mice

4.1 Animals

BALB/c Mice, all of which are male, 6 week old, and purchased from theLaboratory Animal Center of Academy Of Military Medical Sciences,Beijing, were divided into 4 groups as follows with 5 mice in eachgroup: (1) the control group; (2) the Peptide A group, immunized withPeptide A; (3) the Peptide B group, immunized with Peptide B; and (4)the Peptide C group, immunized with Peptide C.

4.2 Method of Immunization

50 μl of the peptide (100 μg/μl) used in the corresponding group and 50μl of Freund's complete adjuvant (GIBCOBRL Corp.) were mixed thoroughlywith a microstirrer, and the mixture was injected into the claw pad of amouse, thereby the first immunization was performed for each of themice. 14 days later, booster immunization was performed in the samemanner as the first immunization with 50 μl of the peptide (100 μg/μl)used in the corresponding group and 50 μl of Freund's complete adjuvant(GIBCOBRL Corp.). 14 days later, another booster immunization wasperformed in the same manner. 14 days later, boost immunization wasperformed by injecting 100 μg of the peptide dissolved in 200 μl PBSinto the mouse intramuscularly. During the process of immunization, onlythe corresponding adjuvant and PBS were injected for the control group.At the first day after the fourth immunization, mice were sacrificed andsera were collected.

4.3 Determination of Levels of Antibodies in Sera of Mice by ELISA Assay

The antibodies in sera of mice were detected by standard indirect ELISAtechnology. The wells of polypropylene/ethylene microtiter plate werecoated with Peptide A, Peptide B, and Peptide C respectively, namely 10μg of the above peptides in 100 μl of basic coating buffer coated eachwell of the plate respectively, and placed at 4° C. overnight. Then theplate was blocked with 1200 PBS, and incubated at 37° C. for 2 hours.Sera (dilution of 1:100) from the mice of each group were then added toeach well, and incubated at 37° C. for 1 hour. 1000 of Goat anti-mouseIgGs (dilution of 1:1000) (purchased from Sino-American BiotechnologyCompany, Beijing) were added to each well, and incubated at 37° C. for 1hour. Then, 50 μl of Solution A and Solution B (purchased from BeijingKewei Reagent Company) were added, and placed in the dark for 5 min,before measuring the optical density (OD) at 450 nm. The results areshown in FIG. 1.

The above results show that all of the mice immunized with the abovepeptides can induce considerable humoral immune response in the bodies.

4.4 Determination of Levels of Cytokines in Sera of Mice by ELISA Assay

According to the specification of the manufacturer, the levels ofcytokines (i.e., IL-4, IL-10, and γ-IFN) in sera of the above immunizedmice were detected respectively by using IL-4 Quantikine ELASA Kit,IL-10 Quantikine ELASA Kit, and γ-IFN Quantikine ELASA Kit (all of whichare purchased from R&D, Inc.). The results are shown in Table 3.

TABLE 3 the Levels of Cytokines in Sera of the Immunized Mice IL-4(pg/mlIL-10(pg/ml γ-IFN(pg/ml Group serum) serum) serum) The control  2.6 ±0.1  5.86 ± 1.0  5.9 ± 0.2 group The Peptide A 29.64 ± 4.9 27.94 ± 6.423.46 ± 5.9 group The Peptide B 30.64 ± 4.9 27.94 ± 6.4 28.46 ± 5.9group The Peptide C 31.22 ± 5.0 26.926 ± 5.8  24.81 ± 5.8 group

The above results show that immunization with the above peptides resultsin a significant increase in γ-IFN, IL-4, and IL-10 in sera of the mice.γ-IFN, a significant cytokine secreted from Th1, is one of importantcytokines against virus infection in immune system of the body, whichcan activate RNase L by inducement of 2-5A synthetase to degrade viralRNA, so as to inhibit the synthesis of viral proteins. Therefore, theincreased level of γ-IFN suggests that the above peptides can be used toeliminate HCV.

Example 5 Effects of the Peptide on γ-IFN of Rats In Vivo

5.1 Animals

SD Rats (body weight of each was 180 g˜220 g; half of them were male andthe others were female; and they were purchased from the AnimalInstitute of Academy Of Military Medical Sciences) were divided randomlyinto 5 groups as follows with 10 rats in each group: (1) the blankcontrol group; (2) the adjuvant control group; (3) the high dose group,immunized with Peptide A and adjuvant; (4) the low dose group, immunizedwith Peptide A and adjuvant; and (5) the peptide group, immunized withPeptide A only.

5.2 Methods

Rats in the high dose group and the low dose group were immunized asfollows: At Day 0, the first immunization was performed by the followingprocess: Peptide A dissolved in physiological saline was mixedthoroughly with the equivalent volume of Freund's complete adjuvant, andthen 0.1 ml of the resultant water-in-oil emulsion was administered viasubcutaneous injection to each rat, wherein the dosage of Peptide Acontained in the emulsion was 50 μg/kg rat body weight for the high dosegroup and 25 μg/kg rat body weight for the low dose group respectively.4 days later, the second immunization was performed in the same dosageand manner as the first immunization except that, Freund's completeadjuvant was replaced by Freund's incomplete adjuvant. 4 days later, thethird immunization was performed in the same dosage and manner as thefirst immunization except that, Freund's complete adjuvant was replacedby Freund's incomplete adjuvant. 4 days later, the fourth immunizationwas performed by the following process: 0.1 ml of Peptide A dissolved inphysiological saline was administered without any adjuvants viaintraperitoneal injection to each rat, wherein the dosage of Peptide Awas 50 μg/kg rat body weight for the high dose group and 25 m/kg ratbody weight for the low dose group respectively. During the process ofimmunization, starting at Day 0, only the corresponding adjuvant andphysiological saline without Peptide A were injected into each rat ofthe adjuvant control group at the days of immunization; and onlyphysiological saline was injected into each rat of the blank controlgroup at the days of immunization.

At the days of immunization for the high dose group and the low dosegroup, starting at Day 0, 0.1 ml of Peptide A dissolved in physiologicalsaline without any adjuvants was administered via subcutaneous injectionto each rat of the peptide group, for immunization up to four times,wherein the dosage of Peptide A was 50 μg/kg rat body weight for everyimmunization.

At the first day after the fourth immunization, rats from every groupwere sacrificed and splenocytes were collected. The splenocytes werethen added into the culture plate at 1×10⁶ cells/well, incubated at 37°C. for 3 days, and then the supernatant of the culture was collected.According to the specification of the manufacturer, the level of γ-IFNin sera of the above immunized rats was detected by using γ-IFN ELASAQuantikine ELASA Kit (purchased from R&D, Inc.).

5.3 Results

The results for the level of γ-IFN in sera of rats are shown in FIG. 2.The results show that when compared to both of the control groups, thereis a significant increase in γ-IFN in sera of the immunized rats. It issurprising that the level of γ-IFN in sera of the rats immunized onlywith Peptide A and with no adjuvant is even higher than that with thesame dosage of Peptide A and adjuvant.

Example 6 Protective Effects of the Peptide on BCG Vaccine andLipopolysaccharide-induced Immunological Liver Damage of Rats

6.1 Animals

Wistar Rats (body weight of each was 180 g˜220 g; half of them were maleand the others were female; and they were purchased from Shanghai SlacLaboratory Animal, Inc.) were divided randomly into 8 groups as followswith 10 rats in each group: (1) the diammonium glycyrrhizinate injectiongroup, injected with diammonium glycyrrhizinate purchased from JiangsuChia-tai Tianqing Pharmaceutical Co. Ltd.; (2) the interferon group,injected with recombinant human interferon-α2a purchased from ShenyangSunshine Pharmaceutical Co., Ltd.; (3) the high dose group, immunizedwith Peptide A; (4) the medium dose group, immunized with Peptide A; (5)the low dose group, immunized with Peptide A; (6) the low dose &adjuvant group, immunized with Peptide A and adjuvant; (7) the blankcontrol group; and (8) the model group.

6.2 Methods

Rats in the low dose & adjuvant group were immunized as follows: At Day0, the first immunization was performed by the following process: 50 μlof Peptide A dissolved in physiological saline was mixed thoroughly withthe equivalent volume of Freund's complete adjuvant, and then 0.1 ml ofthe resultant water-in-oil emulsion was administered via subcutaneousinjection to each rat, wherein the dosage of Peptide A contained in theemulsion was 43.5 μg/kg rat body weight. 4 days later, the secondimmunization was performed in the same dosage and manner as the firstimmunization except that, Freund's complete adjuvant was replaced byFreund's incomplete adjuvant. 4 days later, the third immunization wasperformed in the same dosage and manner as the first immunization exceptthat, Freund's complete adjuvant was replaced by Freund's incompleteadjuvant. 4 days later, the fourth immunization was performed by thefollowing process: 0.1 ml of Peptide A dissolved in physiological salinewas administered without any adjuvants via intraperitoneal injection toeach rat, wherein the dosage of Peptide A was 43.5 μg/kg rat bodyweight.

At the days of immunization for the low dose & adjuvant group, startingat Day 0, 0.1 ml of Peptide A dissolved in physiological saline wasadministered without any adjuvants via subcutaneous injection to eachrat of the high dose group, the medium dose group, and the low dosegroup, for immunization up to four times, wherein for everyimmunization, the dosage of Peptide A was 174, 87, and 43.5 μg/kg ratbody weight respectively for the high dose group, the medium dose group,and the low dose group.

At the days of immunization for the low dose & adjuvant group, startingat Day 0, 0.1 ml of physiological saline was injected into each rat ofthe model group and the blank control group.

And at the 9^(th) day before the fourth immunization, afterPolysaccharide and Nucleic Acid fraction of Bacillus Calmette Guerin forInjection, which was produced by Hunan Jiuzhitang Siqi BiopharmaceuticalCo., Ltd. and comprised 0.35 mg polysaccharide of Bacillus CalmetteGuerin per milliliter, was diluted with physiological saline, 0.2 ml ofthe diluted product was intravenously injected into the tail of each ratof the 7 groups other than the blank control group, at the dosage 126 μgpolysaccharide of Bacillus Calmette Guerin/kg rat body weight;meanwhile, the equivalent volume of physiological saline was injectedfor the blank control group. At the third day after the fourthimmunization, 10 μg of lipopolysaccharide (LPS, available from SIGMA,Inc.) was administered via intravenous injection to each rat of the 7groups other than the blank control group; meanwhile, the equivalentvolume of physiological saline was injected for the blank control group.

In addition, from the 7^(th) day before injecting LPS on, diammoniumglycyrrhizinate was administered via intraperitoneal injection to eachrat of the diammonium glycyrrhizinate injection group every day, at thedosage of 13.5 mg diammonium glycyrrhizinate/kg rat body weight, lastingfor 7 days; and from the 7^(th) day before injecting LPS on, interferonwas administered via intraperitoneal injection to each rat of theinterferon group every day, at the dosage of 540 thousand units/kg ratbody weight, lasting for 7 days.

At the 12^(th) hour after injecting lipopolysaccharide, each rat wasweighed and then sacrificed by cervical dislocation, before blood wascollected. After sera were isolated, according to the specification ofthe manufacturer, the activities of glutamic-pyruvic transaminase (SGPT)and glutamic-oxalacetic transaminase (SGOT) in sera were detectedrespectively by using Glutamic-pyruvic Transaminase Detection Kit(purchased from Nanjing Jiancheng Bioengineering Institute) andGlutamic-oxalacetic Transaminase Detection Kit (purchased from NanjingJiancheng Bioengineering Institute). At the same time, after the liversobtained from the rats were fixed in 10% formalin, and then dehydrated,coated with paraffin, sliced (4 μm thick), and HE stained, theprofessional pathologist examined them under the optical microscope forthe following pathological conditions and scored: (1) degeneration oflive cells, including steatosis, liver edema, acidophilic degenerationand the like; (2) necrosis of liver cells, including spotty necrosis,focal necrosis and the like; (3) dilatation and congestion of centralvein and hepatic sinus, and perivasculitis of liver; (4) connectivetissue proliferation or inflammatory cell infiltration in liver andportal area. According to the severity of each pathological condition,the criteria of pathological condition score are defined as 0 fornormal, 0.5 for extremely minor, 1 for minor, 2 for medium, 3 forsevere, and 4 for extremely severe, and double scored in the presence offocal necrosis. Then the average score was calculated for each group ofanimals, and the more average score represents the worse pathologicalconditions.

6.3 Results

The results of activities of glutamic-pyruvic transaminase (SGPT) andglutamic-oxalacetic transaminase (SGOT) are shown in FIG. 3, and theresults of the pathological condition scores are shown in FIG. 4.

There is a significant increase in glutamic-pyruvic transaminase andglutamic-oxalacetic transaminase in sera of rats in the model group,indicating that BCG vaccine and lipopolysaccharide can causeimmunological liver damage of rats. Peptide A at high, medium, and lowdosage can result in a significant decrease in the levels ofglutamic-pyruvic transaminase and glutamic-oxalacetic transaminase ofrats with liver damage, and the protective effect on acute immunologicalliver damage is also shown in the low dose & adjuvant group. Namely, inthe model of BCG vaccine and lipopolysaccharide-induced immunologicalliver damage of rat, Peptide A can result in a significant decrease inthe levels of glutamic-pyruvic transaminase and glutamic-oxalacetictransaminase of the rat with immunological liver damage, however,diammonium glycyrrhizinate injection has no significant protectiveeffect on liver damage of rats.

The study on pathological histology indicates that the experiment hassuccessfully reproduced the model of immunological liver damage. Thepathological conditions of liver tissues obtained from the model grouphave been shown to include spotty necrosis of liver cells and some focalnecrosis; the increased amount of neutrophilic granulocytes in liver,particularly around central vein; and minor inflammatory cellinfiltration in portal area. The results of pathological condition scoreshow that when compared to the model group, the pathological conditionsof the animals in the high dose group, the medium dose group, the lowdose & adjuvant group and the interferon group have been alleviatedsignificantly (P<0.05). Namely, Peptide A at high and medium dosagewithout any adjuvants and Peptide A at low dosage with adjuvant, canresult in a significant alleviation in pathological condition of liver,however, diammonium glycyrrhizinate injection has no significantprotective effect on liver damage of rats.

Example 7 Protective Effects of the Peptide on D-aminogalactose-inducedAcute Liver Damage of Rats

7.1 Animals

Wistar Rats (body weight of each was 180 g-220 g; half of them were maleand the others were female; and they were purchased from Shanghai SlacLaboratory Animal, Inc.) were divided randomly into 6 groups as followswith 10 rats in each group: (1) the diammonium glycyrrhizinate injectiongroup, injected with diammonium glycyrrhizinate; (2) the high dosegroup, immunized with Peptide A and adjuvant; (3) the medium dose group,immunized with Peptide A and adjuvant; (4) the low dose group, immunizedwith Peptide A and adjuvant; (5) the blank control group; and (6) themodel group.

7.2 Methods

Rats in the high dose group, the medium dose group and the low dosegroup were immunized as follows: At Day 0, the first immunization wasperformed by the following process: 100 μl of Peptide A dissolved inphysiological saline was mixed thoroughly with the equivalent volume ofFreund's complete adjuvant, and then 0.2 ml of the resultantwater-in-oil emulsion was administered via subcutaneous injection toeach rat, wherein the dosage of Peptide A contained in the emulsion was174 μg/kg rat body weight for the high dose group, 87 μg/kg rat bodyweight for the medium dose group, and 43.5 μg/kg rat body weight for thelow dose group respectively. 14 days later, the second immunization wasperformed in the same dosage and manner as the first immunization exceptthat, Freund's complete adjuvant was replaced by Freund's incompleteadjuvant. 14 days later, the third immunization was performed in thesame dosage and manner as the first immunization except that, Freund'scomplete adjuvant was replaced by Freund's incomplete adjuvant. 14 dayslater, the fourth immunization was performed by the following process:0.1 ml of Peptide A dissolved in physiological saline was administeredwithout any adjuvants via intraperitoneal injection to each rat, whereinthe dosage of Peptide A was 174 μg/kg rat body weight for the high dosegroup, 87 μg/kg rat body weight for the medium dose group, and 43.5μg/kg rat body weight for the low dose group respectively.

At the days of immunization for the high dose group, the medium dosegroup and the low dose group, starting at Day 0, the correspondingphysiological saline was injected into each rat of the model group andthe blank control group, for up to four times.

At the 24^(th) hour after the fourth immunization, 0.2 ml ofD-aminogalactose (available from SIGMA, Inc.) was administered viaintraperitoneal injection to each rat of the 5 groups other than theblank control group, at the dosage of 600 mg/kg rat body weight;meanwhile, the equivalent volume of physiological saline was injectedfor the blank control group. At the 48^(th) hour after intraperitoneallyinjecting D-aminogalactose, each rat was sacrificed.

In addition, from the 7^(th) day before sacrificing each rat on,diammonium glycyrrhizinate was administered via intraperitonealinjection to each rat of the diammonium glycyrrhizinate injection groupevery day, at the dosage of 13.5 mg diammonium glycyrrhizinate/kg ratbody weight, lasting for 7 days.

At the 48^(th) hour after intraperitoneally injecting D-aminogalactose,each rat was sacrificed by cervical dislocation, and blood wascollected. After sera were isolated, according to the specification ofthe manufacturer, the activities of glutamic-pyruvic transaminase (SGPT)and glutamic-oxalacetic transaminase (SGOT) in sera were detectedrespectively by using Glutamic-pyruvic Transaminase Detection Kit(purchased from Nanjing Jiancheng Bioengineering Institute) andGlutamic-oxalacetic Transaminase Detection Kit (purchased from NanjingJiancheng Bioengineering Institute).

7.3 Results

The results are shown in FIG. 5. There is a significant increase in thelevels of glutamic-pyruvic transaminase and glutamic-oxalacetictransaminase in sera of rats in the D-aminogalactose model group,indicating that D-aminogalactose can cause acute liver damage of rats.Peptide A at high, medium, and low dosage can result in a significantdecrease in the levels of glutamic-pyruvic transaminase andglutamic-oxalacetic transaminase of rats with acute liver damage in theexperiment, and the diammonium glycyrrhizinate injection is also shownto have the significant protective effect on acute liver damage of rats.

Example 8 Protective Effects of the Peptide onTetrachloromethane-induced Liver Damage of Mice

8.1 Animals

BALB/c mice, each of which is 18 g-22 g weight, and purchased fromShanghai Slac Laboratory Animal, Inc., were divided randomly into 8groups as follows with 10 rats in each group: (1) the diammoniumglycyrrhizinate injection group, injected with diammoniumglycyrrhizinate; (2) the interferon group, injected with recombinanthuman interferon-α2a); (3) the high dose group, immunized with Peptide Aand adjuvant; (4) the medium dose group, immunized with Peptide A andadjuvant; (5) the low dose group, immunized with Peptide A and adjuvant;(6) the adjuvant control group, immunized with adjuvant only; (7) theblank control group; and (8) the model group.

8.2 Methods

At the first before the first immunization, 0.05 ml CCl₄ (available fromShanghai Linfen Chemical Reagent Co., Ltd.) was administered viasubcutaneous injection to each mouse of all groups other than the blankcontrol group; and the equivalent volume of physiological saline wasinjected for the blank control group.

Mice in the high dose group, the medium dose group and the low dosegroup were immunized as follows: the first immunization was performed bythe following process: 1000 of Peptide A dissolved in physiologicalsaline was mixed thoroughly with the equivalent volume of Freund'scomplete adjuvant, and then 0.2 ml of the resultant water-in-oilemulsion was administered via subcutaneous injection to each mouse,wherein the dosage of Peptide A contained in the emulsion was 250 μg/kgmouse body weight for the high dose group, 125 μg/kg mouse body weightfor the medium dose group, and 62.5 μg/kg mouse body weight for the lowdose group respectively. 14 days later, the second immunization wasperformed in the same dosage and manner as the first immunization exceptthat, Freund's complete adjuvant was replaced by Freund's incompleteadjuvant. 14 days later, the third immunization was performed in thesame dosage and manner as the first immunization except that, Freund'scomplete adjuvant was replaced by Freund's incomplete adjuvant. 14 dayslater, the fourth immunization was performed by the following process:0.1 ml of Peptide A dissolved in physiological saline was administeredwithout any adjuvants via intraperitoneal injection to each rat, whereinthe dosage of Peptide A was 250 μg/kg mouse body weight for the highdose group, 125 μg/kg mouse body weight for the medium dose group, and62.5 μg/kg mouse body weight for the low dose group respectively.

At the days of immunization for the high dose group, the medium dosegroup and the low dose group, starting at Day 0, the correspondingphysiological saline was injected into each rat of the model group andthe blank control group, for up to four times; meanwhile, thecorresponding adjuvant and physiological saline were injected for theadjuvant control group, for up to four times.

At the first day before the fourth immunization, 0.05 ml CCl₄ wasadministered via subcutaneous injection to each mouse of all groupsother than the blank control group; and the equivalent volume ofphysiological saline was injected for the blank control group. At thesecond day after the fourth immunization, all of mice were sacrificed.

In addition, from the 7^(th) day before sacrificing each mouse on,diammonium glycyrrhizinate was administered via intraperitonealinjection to each mouse of the diammonium glycyrrhizinate injectiongroup every day, at the dosage of 19.5 mg diammonium glycyrrhizinate/kgmouse body weight, lasting for 7 days; and from the 7^(th) day beforesacrificing each mouse on, interferon was administered viaintraperitoneal injection to each mouse of the interferon group everyday, at the dosage of 750 thousand units/kg mouse body weight, lastingfor 7 days.

Just before sacrificing all mice, each rat was weighed, and thensacrificed by cervical dislocation. Then Blood was collected, beforesera were isolated. According to the specification of the manufacturer,the activities of glutamic-pyruvic transaminase (SGPT) andglutamic-oxalacetic transaminase (SGOT) were detected respectively byusing Glutamic-pyruvic Transaminase Detection Kit (purchased fromNanjing Jiancheng Bioengineering Institute) and Glutamic-oxalaceticTransaminase Detection Kit (purchased from Nanjing JianchengBioengineering Institute). At the same time, after the livers obtainedfrom the mice were fixed in 10% formalin, and then dehydrated, coatedwith paraffin, sliced (4 μm thick), and HE stained, the professionalpathologist examined them under the optical microscope for thepathological conditions and scored in the same manner as Example 6.

8.3 Results

The results of activities of glutamic-pyruvic transaminase (SGPT) andglutamic-oxalacetic transaminase (SGOT) are shown in FIG. 6, and theresults of the pathological condition scores are shown in FIG. 7.

There is a significant increase in the levels of glutamic-pyruvictransaminase and glutamic-oxalacetic transaminase in sera of mice in themodel group, indicating that tetrachloromethane can cause acute liverdamage of mice in the experiment. And Peptide A at high, medium, and lowdosage can result in a significant decrease in the levels ofglutamic-pyruvic transaminase and glutamic-oxalacetic transaminase ofmice with acute liver damage in the experiment. However, interferon andadjuvant have no significant activities to decreasing the level ofaminotransferase.

The study on pathological histology indicates that tetrachloromethanecan effectively cause acute liver damage. The pathological conditions ofliver tissues obtained from the model group have been shown to includespotty necrosis of liver cells or focal necrosis; vasculitis presentedin liver; and minor inflammatory cell infiltration and fibroblastproliferation found in portal area. The results of pathologicalcondition score show that when compared to the model group, thepathological conditions of the liver tissues obtained from the high dosegroup have been alleviated significantly.

In addition, the hepatomegaly of mouse liver is more visible in themouse of the model group than in that of both control groups. Theadministration of Peptide A at the dosage of 250 μg/kg mouse body weightcan result in a significant reduction in such tetrachloromethane-inducedhepatomegaly of mice. However, interferon has no significant activity toreduce tetrachloromethane-induced hepatomegaly of mice.

1-18. (canceled)
 19. A method of treating immunological liver damagecomprising administering to a patient in need thereof a therapeuticallyeffective amount of a peptide having the sequence of Formula I or apharmaceutically acceptable salt or ester thereof,Xaa1-Gln-Xaa2-Xaa3-Thr-Ser-Gly-Xaa4  (Formula I) wherein, Xaa1 isabsent, Ala, Gly, Val, Leu or Ile, Xaa2 is Thr or Ser, Xaa3 is Tyr, Pheor Trp, Xaa4 is absent, Ala, Gly, Val, Leu, Ile or Pro.
 20. A methodaccording to claim 19, wherein Xaa1 is Gly, Xaa2 is Thr, Xaa3 is Tyr,and Xaa4 is absent, Ala or Gly.
 21. A method according to claim 20,wherein Xaa4 is absent.
 22. A method according to claim 19, wherein theimmunological liver damage is not HCV-induced immunological liverdamage.
 23. A method according to claim 22, wherein the immunologicalliver damage is BCG vaccine and lipopolysaccharide-induced immunologicalliver damage.
 24. A method according to claim 22, wherein theimmunological liver damage is D-aminogalactose-induced acuteimmunological liver damage.
 25. A method according to claim 20, whereinthe immunological liver damage is not HCV-induced immunological liverdamage.
 26. A method according to claim 21, wherein the immunologicalliver damage is not HCV-induced immunological liver damage.